Arnd Pralle

Remote control of ion channels and neurons through magnetic-field heating of nanoparticles

Recently, optical stimulation has begun to unravel the neuronal processing that controls certain animal behaviours. However, optical approaches are limited by the inability of visible light to penetrate deep into tissues. Here, we show an approach based on radio-frequency magnetic-field heating of nanoparticles to remotely activate temperature-sensitive cation channels in cells. Superparamagnetic ferrite nanoparticles were targeted to specific proteins on the plasma membrane of cells expressing TRPV1, and heated by a radio-frequency magnetic field. Using fluorophores as molecular thermometers, we show that the induced temperature increase is highly localized. Thermal activation of the channels triggers action potentials in cultured neurons without observable toxic effects. This approach can be adapted to stimulate other cell types and, moreover, may be used to remotely manipulate other cellular machinery for novel therapeutics.

Analysis of a RanGTP-regulated gradient in mitotic somatic cells

The RanGTPase cycle provides directionality to nucleocytoplasmic transport, regulating interactions between cargoes and nuclear transport receptors of the importin-β family. The Ran–importin-β system also functions in mitotic spindle assembly and nuclear pore and nuclear envelope formation. The common principle underlying these diverse functions throughout the cell cycle is thought to be anisotropy of the distribution of RanGTP (the RanGTP gradient), driven by the chromatin-associated guanine nucleotide exchange factor RCC1 (refs 1, 4, 5). However, the existence and function of a RanGTP gradient during mitosis in cells is unclear. Here we examine the Ran–importin-β system in cells by conventional and fluorescence lifetime microscopy using a biosensor, termed Rango, that increases its fluorescence resonance energy transfer signal when released from importin-β by RanGTP. Rango is predominantly free in mitotic cells, but is further liberated around mitotic chromatin. In vitro experiments and modelling show that this localized increase of free cargoes corresponds to changes in RanGTP concentration sufficient to stabilize microtubules in extracts. In cells, the Ran–importin-β–cargo gradient kinetically promotes spindle formation but is largely dispensable once the spindle has been established. Consistent with previous reports, we observe that the Ran system also affects spindle pole formation and chromosome congression in vivo. Our results demonstrate that conserved Ran-regulated pathways are involved in multiple, parallel processes required for spindle function, but that their relative contribution differs in chromatin- versus centrosome/kinetochore-driven spindle assembly systems.

The orientation and molecular movement of a k(+) channel voltage-sensing domain.

Voltage-gated channels operate through the action of a voltage-sensing domain (membrane segments S1-S4) that controls the conformation of gates located in the pore domain (membrane segments S5-S6). Recent structural studies on the bacterial K(v)AP potassium channel have led to a new model of voltage sensing in which S4 lies in the lipid at the channel periphery and moves through the membrane as a unit with a portion of S3. Here we describe accessibility probing and disulfide scanning experiments aimed at determining how well the K(v)AP model describes the Drosophila Shaker potassium channel. We find that the S1-S3 helices have one end that is externally exposed, S3 does not undergo a transmembrane motion, and S4 lies in close apposition to the pore domain in the resting and activated state.

Stable, high‐affinity streptavidin monomer for protein labeling and monovalent biotin detection

The coupling between the quaternary structure, stability and function of streptavidin makes it difficult to engineer a stable, high affinity monomer for biotechnology applications. For example, the binding pocket of streptavidin tetramer is comprised of residues from multiple subunits, which cannot be replicated in a single domain protein. However, rhizavidin from Rhizobium etli was recently shown to bind biotin with high affinity as a dimer without the hydrophobic tryptophan lid donated by an adjacent subunit. In particular, the binding site of rhizavidin uses residues from a single subunit to interact with bound biotin. We therefore postulated that replacing the binding site residues of streptavidin monomer with corresponding rhizavidin residues would lead to the design of a high affinity monomer useful for biotechnology applications. Here, we report the construction and characterization of a structural monomer, mSA, which combines the streptavidin and rhizavidin sequences to achieve optimized biophysical properties. First, the biotin affinity of mSA (Kd = 2.8 nM) is the highest among nontetrameric streptavidin, allowing sensitive monovalent detection of biotinylated ligands. The monomer also has significantly higher stability (Tm = 59.8°C) and solubility than all other previously engineered monomers to ensure the molecule remains folded and functional during its application. Using fluorescence correlation spectroscopy, we show that mSA binds biotinylated targets as a monomer. We also show that the molecule can be used as a genetic tag to introduce biotin binding capability to a heterologous protein. For example, recombinantly fusing the monomer to a cell surface receptor allows direct labeling and imaging of transfected cells using biotinylated fluorophores. A stable and functional streptavidin monomer, such as mSA, should be a useful reagent for designing novel detection systems based on monovalent biotin interaction. Biotechnol. Bioeng. 2013; 110: 57–67.

Engineered Streptavidin Monomer and Dimer with Improved Stability and Function

Although streptavidins high affinity for biotin has made it a widely used and studied binding protein and labeling tool, its tetrameric structure may interfere with some assays. A streptavidin mutant with a simpler quaternary structure would demonstrate a molecular-level understanding of its structural organization and lead to the development of a novel molecular reagent. However, modulating the tetrameric structure without disrupting biotin binding has been extremely difficult. In this study, we describe the design of a stable monomer that binds biotin both in vitro and in vivo. To this end, we constructed and characterized monomers containing rationally designed mutations. The mutations improved the stability of the monomer (increase in T(m) from 31 to 47 °C) as well as its affinity (increase in K(d) from 123 to 38 nM). We also used the stability-improved monomer to construct a dimer consisting of two streptavidin subunits that interact across the dimer-dimer interface, which we call the A/D dimer. The biotin binding pocket is conserved between the tetramer and the A/D dimer, and therefore, the dimer is expected to have a significantly higher affinity than the monomer. The affinity of the dimer (K(d) = 17 nM) is higher than that of the monomer but is still many orders of magnitude lower than that of the wild-type tetramer, which suggests there are other factors important for high-affinity biotin binding. We show that the engineered streptavidin monomer and dimer can selectively bind biotinylated targets in vivo by labeling the cells displaying biotinylated receptors. Therefore, the designed mutants may be useful in novel applications as well as in future studies in elucidating the role of oligomerization in streptavidin function.

Photonic Force Microscopy: A New Tool Providing New Methods to Study Membranes at the Molecular Level

The PFM is a three-dimensional scanning probe microscope based on optical tweezers. It evolved from AFM techniques by replacing the mechanical cantilever of the AFM with optical tweezers and the cantilever tip by a trapped bead. Various detection systems measure the three-dimensional position of the bead with a spatial and temporal resolution in the nanometer and microsecond range, respectively. Due to the small trapping force constants, thermal position fluctuations of the probe are relatively large in comparison to the thermal motion of an AFM cantilever. However, these position fluctuations provide information about the local environment and specific interactions of the probe with molecules such as membrane proteins. Within the few last years, the instrument turned out to be a powerful tool to study properties of the plasma membrane of intact cells at the nanometer scale. For instance, the diffusion of membrane components could be observed over minutes at high spatial and temporal resolutions. For the first time, the diffusion coefficient measured locally in the plasma membrane of an intact cell agreed well with previous measurements for lipid model membranes, thus providing new ways to characterize membrane structures with unknown properties, such as lipid rafts. Furthermore, the technique can be used to determine the elasticity of the lipid bilayer and the binding properties of membrane components to the cytoskeleton.

Compartmentalization of the Cell Membrane.

Many cell-membrane-associated processes require transient spatiotemporal separation of components on scales ranging from a couple of molecules to micrometers in size. Understanding these processes mechanistically involves understanding how lipids and proteins self-organize and interact with the cell cortex. Here, we review recent advances in dissecting the mechanisms of cell membrane compartmentalization. We introduce the challenges in studying cell membrane organization, the current understanding of how complex membranes self-organize to form transient domains, and the role of protein scaffolds in membrane organization. We discuss the formation of signaling domains as an important example of transient membrane compartmentalization. We conclude by pointing to the current limitations of measuring membrane organization in living cells and the steps that are required to advance the field.

Determination and correction of position detection nonlinearity in single particle tracking and three-dimensional scanning probe microscopy.

A general method is presented for determining and correcting nonlinear position detector responses in single particle tracking as used in three-dimensional scanning probe microscopy based on optical tweezers. The method uses locally calculated mean square displacements of a Brownian particle to detect spatial changes in the sensitivity of the detector. The method is applied to an optical tweezers setup, where the position fluctuations of a microsphere within the optical trap are measured by an interferometric detection scheme with nanometer precision and microsecond temporal resolution. Detector sensitivity profiles were measured at arbitrary positions in solution with a resolution of approximately 6 nm and 20 nm in the lateral and axial directions, respectively. Local detector sensitivities are used to reconstruct the real positions of the particle from the measured position signals.

Magnetothermal genetic deep brain stimulation of motor behaviors in awake, freely moving mice

Establishing how neurocircuit activation causes particular behaviors requires modulating the activity of specific neurons. Here, we demonstrate that magnetothermal genetic stimulation provides tetherless deep brain activation sufficient to evoke motor behavior in awake mice. The approach uses alternating magnetic fields to heat superparamagnetic nanoparticles on the neuronal membrane. Neurons, heat-sensitized by expressing TRPV1 are activated with magnetic field application. Magnetothermal genetic stimulation in the motor cortex evoked ambulation, deep brain stimulation in the striatum caused rotation around the body-axis, and stimulation near the ridge between ventral and dorsal striatum caused freezing-of-gait. The duration of the behavior correlated tightly with field application. This approach provides genetically and spatially targetable, repeatable and temporarily precise activation of deep-brain circuits without the need for surgical implantation of any device.

A role for the thermal environment in defining co-stimulation requirements for CD4(+) T cell activation.

Maintenance of normal core body temperature is vigorously defended by long conserved, neurovascular homeostatic mechanisms that assist in heat dissipation during prolonged, heat generating exercise or exposure to warm environments. Moreover, during febrile episodes, body temperature can be significantly elevated for at least several hours at a time. Thus, as blood cells circulate throughout the body, physiologically relevant variations in surrounding tissue temperature can occur; moreover, shifts in core temperature occur during daily circadian cycles. This study has addressed the fundamental question of whether the threshold of stimulation needed to activate lymphocytes is influenced by temperature increases associated with physiologically relevant increases in temperature. We report that the need for co-stimulation of CD4+ T cells via CD28 ligation for the production of IL-2 is significantly reduced when cells are exposed to fever-range temperature. Moreover, even in the presence of sufficient CD28 ligation, provision of extra heat further increases IL-2 production. Additional in vivo and in vitro data (using both thermal and chemical modulation of membrane fluidity) support the hypothesis that the mechanism by which temperature modulates co-stimulation is linked to increases in membrane fluidity and membrane macromolecular clustering in the plasma membrane. Thermally-regulated changes in plasma membrane organization in response to physiological increases in temperature may assist in the geographical control of lymphocyte activation, i.e., stimulating activation in lymph nodes rather than in cooler surface regions, and further, may temporarily and reversibly enable CD4+ T cells to become more quickly and easily activated during times of infection during fever.